momlabfandomcom-20200214-history
RPE-1 cells
Passaging Adherent Cells Materials: DPBS Growth Media 0.25% Trypsin with 2.21 mM EDTA # Aspirate media off cells. # Add enough DPBS to fully cover cells and rotate gently to mix well. 2.1.The amount you will need completely varies on dish size and presence. I usually grow my cells in 100 mm dishes, so I add anywhere from 2-5 ml of DPBS per dish. # Aspirate DPBS # Add Trypsin 4.1.Again, this depends on the size of your dish, for a 100 mm dish I usually add 1 ml, which is enough to cover the surface. # Incubate cells in incubator ~ 5 min. Periodically check cells to see if they have dissociated. More confluent cells will take longer than cells than cells that are sparse. Please keep in mind that you do not want to over trypsinize, this is harmful to the cells. # After cells are trypsinized, add Complete media to resuspend cells. 6.1.Media volume added should be at least 4X the volume of trypsin that was added. 6.2.Example, if you added 1 ml of trypsin, add at least 4 or 5 ml of media to quench the trypsin. # Resuspend cells by gently pipetting up and down. Try not to make bubbles. # Transfer cell suspension to a conical tube and spin down ~ 10 min at low speed about 1,500-2000 x g. 9. Aspirate media and resuspend pellet in complete media. 10. Re-plate desired fraction of cells back into plate. 11. Fill plate back up with complete media and shake plate gently to distribute cells evenly and place back in incubator. 1. The working volume for each plate is different, for a 10 cm dish I usually use ~8-10 ml of media. RPE-1 Cells Growth media: Dulbecco's Modi cation of Eagle's Medium/Ham's F-12 50/50 Mix e formulation contains L-glutamine and 15 mM HEPES We supplement it with 10% FBS and 1X Penicillin/Streptomycin We buy ours from Corning, Cat.# 10-092-CV Synchronization with Ro3306 R&D Systems 4181/10 Fisher Scienti c Cat.# 41-811-0 * Stock solution: 6 mM in DMSO * Working concentration: 6 μM * Make sure cells are not 100% con uent when adding block. Ideally want them between 30-70% so that they have room to divide. * Cells need to block for 18 hours * When releasing cells from block: *# Wash cells 2X with DPBS, and then once with complete media. *# Aspirate last media wash and add appropriate volume of complete media (depending on dish) and allow cells to grow @ 37°C for desired time. Notes: * I personally like to seed the cells a day before (~30-40% con uency) to allow them to stick well to the plate and add the block in the next day. You can also seed in the morning and add the block later in the a ernoon. * Coating coverslips with poly-D-lysine or poly-L-lysine really help cells stick to coverslips. RPE-1 mitotic cells usually stick very well even without the coating. * Below I have attached my most recent optimization for cell cycle arrests at G1, G2 and M phase using Ro3306. RPE-1 Centrin GFP Cell Cycle arrest at G1, G2, and M phase using Ro3306 (CDK1 Inhibitor)-Monday, May 1, 2017 Purpose: Synchronize cells into different stages of the cell cycle using CDK1 inhibitor, RO3306. Will stain for mitotic cells using PH3. Will optimize this to use it to visualize the levels of PCNT mRNA at different cell cycle stages. Cell cycle arrest strategy: Tanenbaum ME, Stern-Ginossar N, Weissman JS, Vale RD. Regulation of mRNA translation during mitosis. Elife (2015) 4.10.7554/eLife.07957 free article PubMed Ref. We will be modifying some of the release time to best t our needs. 2. Seeded RPE-1 Centrin GFP cells into 4 different dishes ~ 35% con uence and allowed to grow O/N. 10 cm Dish Wednesday, May 3, 2017 3. Added Ro3306 inhibitor to cells at different times: 3.1. G1 at 3 PM 3.2. G2/M at 4 PM 3.3. Ro3306 Stock is 6 mM, working concentration is 6 μM (1:1000 dilution). G2 M Phase G1 ursday, May 4, 2017 4. Released cells in 24 well plate # 4.1. G2 arrested cells were xed right a er release ~ 10 AM. # 4.2. M phase cells were xed at 20 min and 30 min a er release ~ 10:30 AM # 4.3. G1 phase: 4.3.1. Cells were released at 9 AM and allowed to grow for 30 min. 4.3.2. Media was then aspirated and plates were shaken with a hard slap or two. 4.3.3.Shaken off cells where then resuspended in more media ( ~ 5 ml in 10 cm and 1 ml per well in 6-well) and pelleted. 4.3.4. Resuspended pellet in fresh media and plated cells onto 3 - 12mm CS in a 24-well plate. 4.3.5. Cells grew for ~3.25 hours and then were xed. Will leave one CS to grow for 6 hrs this time. All cells were xed with 0.5 mL of 4% paraformaldehyde in 1x PBS and incubated for 10 min at RT. 5.1. Processed 2 cover slips for G2 and M phase samples. G1 phase had 3 samples: 2 that attached for 3 hrs and one sample that attached for 6 hours. Block for 1 hr at RT with 250 μl New Block. 6.1. Will block O/N this time 5. 6. Friday, May 5, 2017 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. Add appropriately diluted primary antibody in 1% BSA in 1X PBS 7.1. Added 1 μl of Rabbit-anti-PH3 in 500 μl of 1 % BSA in 1X PBS. (1:500 dilution) 7.2. Added 67 μl to each coverslip Incubate for 1 hr at RT. Washed 3x quickly with 1X PBS. Washed 4x for 5 min with 1X PBS. Add appropriately diluted Secondary antibody with 1% BSA in 1X PBS. 11.1.Secondary antibody mix for samples had 1 μl of Goat-anti-Rabbit AF 568 in 1% BSA in 1X PBS. (1:500 dilution) 11.2.Added 67 μl to each coverslip. Incubate at for 1 hr at RT. Washed 3x quickly with 1X PBS. Washed 4x for 5 min with 1X PBS. Stain with 0.05μg/mL DAPI diluted in 1x PBS (1:2000 dilution of 100 μg/ml) for 30 min. Mount slide with 8 μl of Vectashield per coverslip. Allowed them to dry for a minimum of 30 min before taking a quick look on Microscope Seal CS with nail polish a er mounting media has dried. Results: 6 hours a er repeating worked best for G1, 20 min release worked best for M phase. ~70% of cells are synchronized into G2 when xing right a er release.